The term “bubble” as used herein refers to a bubble of gas encased or surrounded by an enclosing substance. Bubbles that are from one micrometer to several tens or hundreds of micrometers in size are commonly referred to as “microbubbles”, while bubbles that are smaller than one micrometer in size are commonly referred to as “nanobubbles.” The term “droplet” as used herein refers to an amount of liquid that is encased or surrounded by a different, enclosing substance. Droplets that are less than one micrometer in size are commonly referred to as “nanodroplets” and those that are in the one micrometer to tens or hundreds of micrometers in size are commonly referred to as “microdroplets.” If a droplet is encased in another liquid, the droplet and its casing may also be referred to as an “emulsion”. The term “particle” as used herein refers to either a droplet or a bubble of any size.
Microbubbles for diagnostic ultrasound imaging have been established in the clinical arena as a sensitive and inexpensive imaging technique for interrogating landmarks in the vasculature. Currently, microbubble-enhanced diagnostic ultrasound has been approved by the FDA for the study of wall motion abnormalities and ventricular contraction in echocardiography. Researchers have proposed microbubble-aided ultrasound for a wide range of potential applications, including functional tumor, kidney, and liver imaging, identification of vascular inflammation, identification of vulnerable plaque deposition, thrombus detection and targeted molecular imaging of angiogenesis. Microbubbles have been used for therapeutic interventions, primarily in concert with ultrasound-mediated cavitation for sonothrombolysis.
Despite their utility as vascular contrast agents and potential for therapeutic applications, microbubble size (typically 1-5 microns in diameter) prevents their transport outside of the vasculature, a process commonly referred to as extravasation. In other words, microbubbles are trapped within the circulatory system. In order to extravasate into the interstitial space in a solid tumor, the bubble would need to be smaller than a micron, i.e., a nanoparticle is required. The exact size limit for nanoparticle extravasation into the interstitial space in solid tumors depends on a variety of factors, but has been reported to fall within the range of 100 nm-750 nm.
Nanoparticles make poor ultrasound contrast agents, however. Nanobubbles small enough to diffuse past inter-endothelial gap junctions scatter ultrasound energy poorly compared to microbubbles and thus provide limited imaging contrast. Additionally, bubble circulation in vivo is shown to be on the order of tens of minutes before bubble dissolution, and clearance significantly limits contrast enhancement. This short time period may be insufficient for enough bubbles to accumulate by diffusion into the tumor interstitium. Droplets of any size provide poor contrast for ultrasound imaging as compared to equivalently sized bubbles, and nanodroplets small enough to extravasate into the interstitial space in solid tumors provide poorer contrast still.
One approach to solve the problem of providing ultrasound contrast agents that are both small enough to extravasate and large enough to provide sufficient ultrasound contrast has been to produce a droplet that is small enough to extravasate but which can be caused to expand into a bubble, a processed referred to as “activation”. Such particles are commonly referred to as “phase change agents”. One method of activation is known as acoustic droplet vaporization, or ADV. In ADV, the droplet is subjected to ultrasonic energy, which causes the liquid within the droplet to change phase and become a gas. This causes the droplet to become a bubble, with the corresponding increase in size. The ultrasound impulses impart a mechanical pressure upon the tissues, and the amount of pressure applied is indicated in terms of a mechanical index, or MI.
Particles that start as droplets but can be activated to become bubbles are referred to as “metastable”, because they are stable as droplets (e.g., they don't spontaneously expand into bubbles) without additional energy. If these PCAs are used as contrast agents, they are commonly referred to as “phase-change contrast agents” (PCCAs).
Recently there has been interest in the use of PFC droplets for this purpose. To date, PCAs have been developed using PFCs which have boiling points above room temperature (25° C.), which are herein referred to as “low volatility PFCs”. Examples include dodecafluoropentane (DDFP), perfluorohexane (PFH), and perfluoroheptane. These low volatility PFCs have been used to make PCAs that have a diameter greater than 1 micron, i.e., microdroplets or microbubbles. PFCs with boiling points above room temperature, which are herein referred to as “high volatility” or “highly volatile” PFCs, have not been used to make microparticles out of a concern that, if subjected to body temperature (37° C.), a droplet containing a highly volatile PFC might spontaneously change phase.
However, the low-volatility PFCs conventionally used to make micro-PCAs are not suitable for making nano-PCAs. Many in vitro studies have shown that the energy required to activate a PFC-based PCA increases as the diameter of the initial droplet decreases. There is a direct correlation between activation energy and mechanical index, and applications involving relatively low frequencies and/or sub-micron droplets may require pressures higher than diagnostic ultrasound machines currently provide. This is an obstacle to human treatment, because excessive ultrasonic activation energy can cause tissue damage or other unwanted bioeffects.
Thus, PFCs that had been used in microbubbles may be unsuitable for use in nanodroplets due to the excessive activation energy required. The smaller the nanodroplet, the more activation energy is required, and the less suitable the PFC. For example, the Antoine vapor pressure equation was analyzed in order to assess the theoretical vaporization temperature dependence upon droplet diameter of selected PFCs as a result of the influence of interfacial surface tension. Using this model to investigate the influence of PFC boiling points, it was concluded that DDFP, PFH, and perfluoroheptane may require a relatively large amount of energy in order to elicit droplet vaporization at a size that would practically be able to extravasate through endothelial gap junctions and into the extravascular space.
Therefore, there exists a need for a phase-change agent that is stable at physiological temperatures yet is more susceptible to ultrasound pressures. Such a particle could provide a more efficacious vehicle for extravasation into tissue and activation at the site of action in many applications. For human therapeutic and diagnostic use, there is a need for a stable nanoparticle capable of being vaporized using frequencies and mechanical indices within the FDA-approved limits of commercial clinical diagnostic ultrasound machines.